Thiol-based redox regulation has emerged as the prevalent mechanism for redox regulation of cellular processes. Its major component, the thioredoxin (Trx) system, is present in all organisms. Thioredoxin reductases (TRs) control the redox state of Trxs, which in turn regulate cellular processes by controlling the redox state of Trxs. TRs and Trxs have been implicated in cancer development, apoptosis, signal transduction and many other processes, but the pathways and mechanisms of thiol-based regulation are poorly understood. Mammals have three TRs: cytosolic TR1 (Txnrd1), mitochondrial TR3 (Txnrd2) and thioredoxin/glutathione reductase TGR (Txnrd3). All three contain a catalytic selenocysteine in the C-terminal region and, therefore, the mammalian Trx system is dependent on dietary selenium. In addition, each TR has several isoforms that differ by their N-terminal sequences. Among TR substrates, previous studies focused on Trx1 and few other thiol oxidoreductases (i.e., proteins that use catalytic redox Cys). However, there are numerous other Trx-like proteins and thiol oxidoreductases, and the overall composition of this system and identity of proteins regulated by thiol oxidoreductases is not known. This is important because thiol oxidoreductases provide a framework for redox regulation of cellular processes by targeting redox Cys residues in proteins. Previously, we defined the basic properties of TRs and their roles in various biological processes, organs and organisms. Additional data revealed successful application of mechanism-based approaches to identify substrates of TRs and thiol oxidoreductases. Computational approaches were also developed that allow identification of entire sets of thiol oxidoreductases in organisms. We now propose to use this foundation to characterize isoforms of TRs, their thiol oxidoreductase substrates and protein targets of thiol oxidoreductases to define the major pathway of thiol-based redox regulation in mammals. To address this challenge, we propose the following specific aims: (1) Characterize regulation of TR function and redox homeostasis by heterodimerization and position-specific Sec insertion. (2) Determine specific substrates of mammalian TR isoforms. (3) Define a mammalian set of thiol oxidoreductases and their protein targets. A combination of computational, proteomic, cell biology, and animal model approaches will be used to address these questions, and the data will be used to define the network of thiol-based redox regulation of protein function in mammals.